There are many instances whereby the efficacy of a therapeutic protein is limited by an unwanted immune reaction to the therapeutic protein. Several mouse monoclonal antibodies have shown promise as therapies in a number of human disease settings but in certain cases have failed due to the induction of significant degrees of a human anti-murine antibody (HAMA) response [Schroff, R. W. et al (1985) Cancer Res. 45: 879-885; Shawler, D. L. et al (1985) J. Immunol. 135: 1530-1535]. For monoclonal antibodies, a number of techniques have been developed in attempt to reduce the HAMA response [WOA8909622; EPA0239400; EPA0438310; WOA9106667; EPA0699755]. These recombinant DNA approaches have generally reduced the mouse genetic information in the final antibody construct whilst increasing the human genetic information in the final construct. Notwithstanding, the resultant “humanized” antibodies have, in several cases, still elicited an immune response in patients [Issacs J. D. (1990) Sem. Immunol. 2: 449, 456; Rebello, P. R. et al (1999) Transplantation 68: 1417-1420].
Antibodies are not the only class of polypeptide molecule administered as a therapeutic agent against which an immune response may be mounted. Even proteins of human origin and with the same amino acid sequences as occur within humans can still induce an immune response in humans. Notable examples include therapeutic use of granulocyte-macrophage colony stimulating factor [Wadhwa, M. et al (1999) Clin. Cancer Res. 5: 1353-1361] and interferon alpha 2 [Russo, D. et al (1996) Bri. J. Haem. 94: 300-305; Stein, R. et al (1988) New Engl. J. Med. 318: 1409-1413] amongst others.
Key to the induction of an immune response is the presence within the protein of peptides that can stimulate the activity of T-cells via presentation on MHC class II molecules, so-called “T-cell epitopes”. Such T-cell epitopes are commonly defined as any amino acid residue sequence with the ability to bind to MHC Class II molecules. Implicitly, a “T-cell epitope” means an epitope which when bound to MHC molecules can be recognized by a T-cell receptor (TCR), and which can, at least in principle, cause the activation of these T-cells by engaging a TCR to promote a T-cell response.
MHC Class II molecules are a group of highly polymorphic proteins which play a central role in helper T-cell selection and activation. The human leukocyte antigen group DR (HLA-DR) are the predominant isotype of this group of proteins however, isotypes HLA-DQ and HLA-DP perform similar functions. The present invention is applicable to the detection of T-cell epitopes presented within the context of DR, DP or DQ MHC Class II. In the human population, individuals bear two to four DR alleles, two DQ and two DP alleles. The structure of a number of DR molecules has been solved and these appear as an open-ended peptide binding groove with a number of hydrophobic pockets which engage hydrophobic residues (pocket residues) of the peptide [Brown et al Nature (1993) 364: 33: Stem et al (1994) Nature 368: 215]. Polymorphism identifying the different allotypes of class H molecule contributes to a wide diversity of different binding surfaces for peptides within the peptide binding grove and at the population level ensures maximal flexibility with regard to the ability to recognize foreign proteins and mount an immune response to pathogenic organisms.
An immune response to a therapeutic protein proceeds via the MHC class II peptide presentation pathway. Here exogenous proteins are engulfed and processed for presentation in association with MHC class II molecules of the DR, DQ or DP type. MHC Class II molecules are expressed by professional antigen presenting cells (APCs), such as macrophages and dendritic cells amongst others. Engagement of a MHC class II peptide complex by a cognate T-cell receptor on the surface of the T-cell, together with the cross-binding of certain other co-receptors such as the CD4 molecule, can induce an activated state within the T-cell. Activation leads to the release of cytokines further activating other lymphocytes such as B cells to produce antibodies or activating T killer cells as a full cellular immune response.
T-cell epitope identification is the first step to epitope elimination, however there are few clear cases in the art where epitope identification and epitope removal are integrated into a single scheme. Thus WO98/52976 and WO00/34317 teach computational threading approaches to identifying polypeptide sequences with the potential to bind a sub-set of human MHC class II DR allotypes. In these teachings, predicted T-cell epitopes are removed by the use of judicious amino acid substitution within the protein of interest. However with this scheme and other computationally based procedures for epitope identification [Godkin, A. J. et al (1998) J. Immunol. 161: 850-858; Sturniolo, T. et al (1999) Nat. Biotechnol. 17: 555-561], peptides predicted to be able to bind MHC class II molecules may not function as T-cell epitopes in all situations, particularly, in vivo due to the processing pathways or other phenomena. In addition, the computational approaches to T-cell epitope prediction have in general not been capable of predicting epitopes with DP or DQ restriction.
Besides computational techniques, there are in vitro methods for measuring the ability of synthetic peptides to bind MHC class II molecules. An exemplary method uses B-cell lines of defined MHC allotype as a source of MHC class II binding surface and may be applied to MHC class II ligand identification [Marshall K. W. et al. (1994) J. Immunol. 152:4946-4956; O'Sullivan et al (1990) J. Immunol. 145: 1799-1808; Robadey C. et al (1997) J. Immunol 159: 3238-3246]. However, such techniques are not adapted for the screening multiple potential epitopes to a wide diversity of MHC allotypes, nor can they confirm the ability of a binding peptide to function as a T-cell epitope.
Recently techniques exploiting soluble complexes of recombinant MHC molecules in combination with synthetic peptides have come into use [Kern, F. et al (1998) Nature Medicine 4:975-978; Kwok, W. W. et al (2001) TRENDS in Immunol. 22:583-588]. These reagents and procedures are used to identify the presence of T-cell clones from peripheral blood samples from human or experimental animal subjects that are able to bind particular MHC-peptide complexes and are not adapted for the screening multiple potential epitopes to a wide diversity of MHC allotypes.
Biological assays of T-cell activation can provide a practical option to providing a reading of the ability of a test peptide/protein sequence to evoke an immune response. Examples of this kind of approach include the work of Petra et al using T-cell proliferation assays to the bacterial protein staphylokinase, followed by epitope mapping using synthetic peptides to stimulate T-cell lines [Petra, A. M. et al (2002) J. Immunol. 168: 155-161]. Similarly, T-cell proliferation assays using synthetic peptides of the tetanus toxin protein have resulted in definition of immunodominant epitope regions of the toxin [Reece J. C. et al (1993) J. Immunol. 151: 6175-6184]. WO99/53038 discloses an approach whereby T-cell epitopes in a test protein may be determined using isolated sub-sets of human immune cells, promoting their differentiation in vitro and culture of the cells in the presence of synthetic peptides of interest and measurement of any induced proliferation in the cultured T-cells. The same technique is also described by Stickler et al [Stickler, M. M. et al (2000) J. Immunotherapy 23:654-660], where in both instances the method is applied to the detection of T-cell epitopes within bacterial subtilisin. Such a technique requires careful application of cell isolation techniques and cell culture with multiple cytokine supplements to obtain the desired immune cell sub-sets (dendritic cells, CD4+ and or CD8+T-cells) and is not conducive to rapid through-put screening using multiple donor samples.
As depicted above and as consequence thereof, it would be desirable to identify and to remove or at least to reduce T-cell epitopes from a given in principal therapeutically valuable but originally immunogenic peptide, polypeptide or protein. One of these therapeutically valuable molecules is a monoclonal antibody with binding specificity for tumor necrosis factor alpha (TNF alpha). The preferred monoclonal antibody of the present invention is a modified form of the antibody cA2 described in U.S. Pat. No. 6,284,471. The antibody cA2 is herein referred to as the “parental” antibody of the invention.
It is an objective of the invention to provide for modified forms of the parental mouse-derived monoclonal antibody with binding specificity to human TNF alpha with one or more T cell epitopes removed. TNF alpha is involved in the mediation of a number of pathological conditions including Crohn's disease, rheumatoid arthritis and endotoxic or cardiovascular shock. The modified antibody of the invention can be expected to have therapeutic utility in these conditions and other diseases in which TNF alpha is a significant component of the pathophysiology.
The cA2 antibody is not the only antibody with binding specificity for TNF alpha. A variety of polyclonal and monoclonal preparations with similar specificity are known in the art. Examples include the anti-TNF alpha preparations disclosed in EP0212489, EP0218868, EP0288088 and WO91/02078. Further examples of rodent or murine monoclonal antibodies specific for recombinant human TNF alpha have been described in the literature [see for example; Liang, et al (1986) Biochem. Biophys. Res. Comm. 137: 847-854; Meager, et al. (1987), Hybridoma 6: 305-311; Fendly et al. (1987), Hybridoma 6: 359-369; Bringman, et al (1987), Hybridoma 6: 489-507; Hirai, et al., (1987) J. Immunol. Meth. 96: 57-62 and Moller, et al (1990), Cytokine 2: 162-169]. Some of these antibodies are able to neutralise the effect of TNF alpha in vitro and have been used to develop immunoassays for TNF alpha or are used in the purification of recombinant TNF alpha. In general, and in contrast with antibody cA2, these antibodies have not been developed for in vivo diagnostic or therapeutic uses in humans.
Clinical studies have however been conducted using a murine anti-TNF alpha antibody in human subjects. In fourteen patients with severe septic shock receiving a single dose of a murine anti-TNF alpha antibody seven developed a human anti-murine antibody response to the treatment, due to the immunogenicity of the therapeutic murine antibody. [Exley, A. R. et al.(1990), Lancet 335: 1275-1277]. Such immunogenicity can render treatment ineffective in patients undergoing diagnostic or therapeutic administration of murine anti-TNF alpha antibodies.
In this regard, Adair et al [EP0927758] describe recombinant antibody molecules including antibodies with human constant region sequences and versions in which the complementarity determining regions (CDRs) have been engrafted onto modified human antibody framework sequences. Such antibodies and humanized antibodies are claimed to retain specificity to human TNF alpha□ and may be used in diagnosis and therapy.
The clinical use of antibody cA2 is described by Le et al [U.S. Pat. No. 5,919,425] who detail methods of treating TNF alpha mediated disease using cA2 which is in fact a chimeric form of an original murine monoclonal antibody designated A2. Similarly, Feldmen et al [U.S. Pat. No. 6,270,766] describe use of the same antibody in combination therapy with a myeloablative agent methotrexate for the treatment of arthritis and Crohn's disease.
Large clinical trials have now been conducted using this antibody, which has received the compound name “infliximab”, and is traded in some territories as REMICADE. This antibody has demonstrated a degree of therapeutic efficacy in the treatment of rheumatoid arthritis and Crohn's disease has received regulatory approval for its use as in Crohn's disease in the USA and in Europe. The antibody is produced by recombinant techniques and as noted above, the antibody is “chimeric” meaning that the constant region of the antibody is comprised of sequence derived from human constant region genes and the contribution of mouse derived protein sequence is therefore reduced. Despite this up to 13% of patients treated for Crohn's disease showed an immune response to the therapeutic antibody [Mani R. N. et al. (1998) Arthritis Rheum. 41:1552-1563; Elliot M. J. et al (1994) Lancet 344: 1105-1110; Targan S. R. et al (1997) N. Engl. J. Med. 337: 1029-1035; Present D. H. et al (1999) N. Engl. J. Med. 340: 1398-1405].
There is therefore a need for anti-TNF alpha antibody analogues with enhanced properties and especially improvements in the biological properties of the protein. In this regard, it is highly desired to provide anti-TNF alpha antibodies with reduced or absent potential to induce an immune response in the human subject. Such proteins would expect to display an increased circulation time within the human subject and would be of particular benefit in chronic or recurring disease settings such as is the case for a number of indications for anti-TNF alpha antibodies. The present invention provides for modified forms of an anti-TNF alpha antibody that are expected to display enhanced properties in vivo.
It is a particular objective of the present invention to provide modified anti-TNF alpha antibodies in which the immune characteristic is modified by means of reduced numbers of potential T-cell epitopes.
The invention discloses sequences identified within the variable region sequences of the heavy and light chains of an anti-TNF alpha antibody that are potential T cell epitopes by virtue of MHC class II binding potential.
The invention further discloses sequences identified within the variable region sequences of the heavy and light chains of an anti-TNF alpha antibody that are potential T cell epitopes by virtue of their ability when presented as synthetic peptides to a population of human peripheral blood mononuclear cells (PBMC) cultured in vitro to induce a proliferative response in said PBMC cells. Such information thereby has enabled the constitution of a map of T-cell epitopes present in the said variable regions of the antibody and has provided the critical information required for removing the T-cell epitopes from the molecule.
Where others have provided anti-TNF alpha antibody molecules including chimeric [U.S. Pat. No. 5,919,425] and humanized [EP0927758] forms, none of these teachings recognize the importance of T cell epitopes to the immunogenic properties of the protein nor have been conceived to directly influence said properties in a specific and controlled way according to the scheme of the present invention.